在HER2过表达乳腺癌细胞中曲妥珠单抗抵抗的分子机制

The molecular mechanism of Trastuzumab resistance in Her2 overexpressing breast cancer cells

目的已有研究显示HER2基因过表达乳腺癌细胞对曲妥珠单抗(TZB)产生抵抗性与LC3的过表达相关,本研究通过RNAi沉默耐TZB的SKBR3(SKBR3-TR)细胞LC3的表达,探讨HER2阳性乳腺癌对TZB抵抗的机制。方法以SKBR3乳腺癌细胞及SKBR3-TR细胞为研究对象,检测两组细胞中LC3表达情况。以SKBR3-TR细胞为研究对象,通过慢病毒载体沉默SKBR3-TR细胞LC3的表达,实时定量PCR(Realtime PCR)、蛋白免疫印迹检测LC3mRNA、蛋白的表达及水平,通过CCK-8法检测基因沉默前后细胞存活率。结果 SKBR3-TR细胞导入RNAiLC3载体后,LC3mRNA及蛋白表达水平呈现明显降低,CCK8法检测显示沉默组细胞存活率明显降低。结论 LC3在HER2阳性乳腺癌对曲妥珠单抗抵抗过程中发挥重要作用,而LC3是自噬的重要标志,说明自噬在引起曲妥珠单抗抵抗HER2阳性乳腺癌中可能发挥某种重要的调控机制。

Objective It has been reported that resistance to the treatment with Trastuzumab in the in breast cancer cells,overexpressing Her2 gene,is associated with the overexpression of LC3.We explored the underlying Trastuzumab resistance mechanism of HER2 positive breast cancer cells through knocking down LC3 expression in SKBR3 Cells by using RNAi technology.Methods Wild type SKBR3 breast cancer cells and Trastuzumab resistant SKBR3 Cells were used as the research object.In both cell lines,LC3 protein level was examined.The expression of LC3 in Trastuzumab resistant SKBR3 Cells was suppressed by Lentiviral vectors.The mRNA and protein expression level of LC3 were examined by Real time PCR and western blotting respectively.The cell survival was examined by CCK-8 before and after LC3 knockdown.Results After Trastuzumab resistant SKBR3 Cells were transfected with the RNAi LC3 vector,The mRNA and protein expression level of LC3 were all downregulated significantly.Importantly,the survival fraction of LC3 knockdown group decreased significantly.Conclusion LC3 plays an important role in the resistance of HER2 positive breast cancer to Trastuzumab.Since LC3 is a well known marker of autophagy,therefore our findings indicate that autophagy plays pro-survival role and augments the resistance of HER2 positive breast cancer cells to Trastuzumab.